Aim: To analyze the bcl-2 protein expression in the epithelial lining of the Odontogenic keratocyst (OKC), Radicular (RC), and Dentigerous cysts (DC).Materials and Methods: Forty-five archival samples of paraffin-embedded tissue sections were utilized. Fifteen OKCs, 15 DC, and 15 RC were immunohistochemically analyzed for immunoreactivity of the bcl-2 protein.Results: Expression of bcl-2 was seen in the basal layer of the epithelial lining of the OKC. DC and RC showed almost complete negativity for bcl-2. There was a statistically significant increased expression in all OKCs.Conclusion: The bcl-2 protein overexpression could increase the survival of the epithelial cells and this increased lifespan could in turn, lead to the peculiar aggressive growth pattern of OKC. However, the bcl-2 staining can be useful to differentiate OKC from other odontogenic cysts.

Kramer has defined a cyst as 'a pathological cavity having fluid, semifluid or gaseous contents and which is not created by accumulation of pus'. Most cysts, but not all, are lined by epithelium. [1]

Odontogenic cysts are those which arise from odontogenic epithelium either from a dental lamina or from the enamel organ of tooth or their remnants. It is usually considered that proliferation and cystic degeneration of this epithelium leads to odontogenic cyst developmemt. [1] Odontogenic keratocysts (OKC) are developmental epithelial cysts, considered to arise from derivatives of dental lamina, or its remnants which normally differentiate into tooth buds and enamel-producing cells and extensions of basal cells from the overlying epithelium. [2],[3] These lesions present an aggressive clinical course frequently characterized by recurrences. [4] Some mechanisms have been suggested to explain its behavior and high tendency for recurrence such as the difficulty to remove it in one piece due to thin friable nature of the cyst wall, satellite small daughter cysts, production of bone resorptive factors in the cyst wall, and increased proliferation of the epithelial lining of the cyst. [5] Several studies were conducted on odontotogenic cysts using proliferative and apoptotic markers such as p53, Ki-67, and bcl-2. OKC showed an increased expression of p53, Ki-67, and bcl-2 in its epithelial lining as compared than other odontogenic cysts. [6]

Apoptotic reactions play various roles in the organization of normal tissues and their pathogenesis by modulation of several proteins. The bcl-2 family is a group of closely related proteins that plays a major role in apoptosis regulation. [7] This is constituted by inductive (e.g., bax) and inhibitory (e.g., bcl-2) apoptotic factors, and cell survival is warranted by higher inhibitory apoptotic gene expression. The bcl-2 protein is a 26-KDa putative member which acts as a cell death suppressor thus facilitating cell survival by regulating apoptosis. [8]

Accumulation of cells with an aberrant bcl-2 expression could be an important step in carcinogenesis bcl-2 expression could probably be related to loss of cell differentiation. Immunoreactivity for bcl-2 family proteins has been detected in odontogenic epithelium under various conditions, suggesting that these proteins play a role in regulating cellular kinetics of odontogenic epithelium. [9],[10] The aim of the present study was to analyze bcl-2 protein expression in OKC as compared to radicular (RC) and dentigerous cysts (DC).

Materials and Methods

The present study was carried out using 45 formalin-fixed, paraffin-embedded blocks diagnosed histologically as OKC, RC, and DC from 2006 to 2010 which were retrieved from the archives of Department of Oral and Maxillofacial Pathology.

The study contained 45 samples of which:

Group A - Odontogenic keratocysts (15),

Group B - Radicular cysts (15)

Group C - Dentigerous cysts (15)

Serial sections of 4 μm were obtained from the archival material. The sections of group A, B, and C were first subjected for routine hematoxylin and eosin examination in order to confirm the diagnosis. Later other sections of all the three groups were subjected for immunohistochemical analysis using monoclonal bcl-2 antibody (Dakopats, CA, USA). The technique used was based on the labeled streptavidin-biotin method. After routine processing of the specimen, the endogenous peroxidase activity of the tissue was blocked by using 3% hydrogen peroxide for 5 mins. The specimen was then incubated with an appropriate primary antibody. The primary antibody used was monoclonal bcl-2. After this step, the non-specific binding sites were blocked with non-immune goat serum. Biotinylated secondary antibody and streptavidin-peroxidase conjugate were added sequentially and the specimen was incubated for 30 mins in each of them. Finally the chromogen diluted in the substrate buffer was added.

A routine processing of all the specimens was done with the help of alcohol, xylene, and finally embedded in paraffin. Sectioning was done using rotary microtome and section of 4-μm thick were cut and carefully fixed on the prepared microslides coated with poly-L-Lysine. The tissue was de-paraffinized by giving two dips, each dip of about 5 mins in fresh xylene. Rehydration of tissue was carried out by giving two dips for 3 mins each in absolute ethanol and it was placed in distilled water bath and not allowed to be dried.

All the reagents were spread evenly on the glass slides by covering the whole tissue section without any air bubble. All reagents are brought to the room temperature and incubation was done at room temperature in a moist chamber.

Positive bcl-2 expression was seen as light brown, granular stain in the cytoplasm of the cell. All stained areas demonstrating positivity for bcl-2 were identified at a magnification of X40 (MAGNUS) and the number of positively stained cells were counted on 10 representative areas of the epithelium using a X40 objective in a minimum of 100 cells in the full length of the epithelium. The intensity of bcl-2 positivity was estimated as:

(-) fewer than 5% positive cells or no staining;

(+) 5-9% positive

(++) 10-24% positive

(+++) 25-50% positive

(++++) more than 50% positive

Collected data were analyzed using the Statistical Package for Social Sciences software. Data analysis was performed using Kruskal-Wallis one-way Anova0 and Mann-Whitney U test with the level of significance set at P < 0.05.

Results

The bcl-2 immunoreactive positive basal cells of the cystic lining were counted manually under the binocular microscope. Expression of bcl-2 in OKC, DC, and RC is shown in [Table 1].

Positive bcl-2 staining was noted in the cytoplasm of the basal cells throughout the epithelium in all most all the cases of OKC, [Figure 1] maximum of about seven cases between the range of 25% and 50%. Only two cases of DC showed positivity for bcl-2 immunoreactivity [Figure 2] between the ranges of 25% and 50%. Almost in nine cases of RC the bcl-2 immunoreactivity was less than 5% [Figure 3].

Significant difference was observed in staining intensity among OKC, DC, and RC using Kruskal-Wallis one-way ANOVA [Table 2]. Pairwise comparison was done between the expression of bcl-2 in all OKC, DC, and RC which revealed a statistically significant difference between OKCs and RCs and between OKCs and DCs using Mann-Whitney U test (P-value < 0.01). But there was no significant difference between RC and DCs in the expression of bcl-2 immunoreactivity which was shown by Mann-Whitney U test (P-value = 0.1585) [Table 3].

OKCs develop from the odontogenic epithelium or its remnants. [11] It is well known that the OKCs show an aggressive behavior, recurring at greater frequency than other types of odontogenic cysts. [12] These clinical findings have been supported by numerous reports focusing on the greater proliferative potential of the epithelial lining of OKCs compared with other types of odontogenic cysts.[13] Consequently, it has been proposed that OKCs should be regarded as benign neoplasms. [5] In relation to the proliferative activity of the lining epithelium of OKCs, a number of investigators have examined the expression of p53 [14], Proliferating Cell Nuclear Antigen (PCNA) and Ki67 in OKCs. Those studies concluded that p53, PCNA, and Ki67 are expressed more strongly in OKCs than in other types of odontogenic cysts. [5]

The role played by apoptosis in the pathogenesis of cysts in the jaws has not been extensively investigated. The apoptotic outcome of the cell is largely determined by the interactions among the bcl-2 family members. [15] Bcl-2 protects cells by blocking post-mitotic differentiation from apoptosis, thus maintaining a stem cell pool. [16] Many researchers have investigated possible differences in the expression of Bcl-2 family between OKCs and other types of odontogenic cysts. [17]

Piattelli [18]et al., in 1998 examined the immunoreactivity of bcl-2 in OKC, DC, and RCs, where the immunoreactivity strikingly showed significant differences between OKC and other types of odontogenic cysts. OKC showed strong bcl-2 positivity in the basal cell layer with a greater than 50% positivity of the cells examined, while, on the contrary, the other types of cysts showed almost complete bcl-2 negativity. Only in one case of dentigerous cyst it was possible to observe few positive cells in the basal and parabasal layers (> 10%).

Lo Muzio [19] in 1999 compared the expression of bcl-2 in 16 cases of sporadic and syndromic cases of OKC each, of which 13 cases showed positivity for bcl-2 and in syndromic cases only one case did not show the expression. None of our cases were syndromic but these findings are also in accordance with our results.

Gh. Jahanshahi [20]et al., in 2005 observed significant difference in staining intensity between OKC, DC, and RC. In multiple comparisons, bcl-2 staining intensity revealed a statistically significant difference between OKCs and RCs and between OKC and DCs. There was no significant difference between RC and DCs.

Similar results were observed in the present study which showed positive bcl-2 staining of the basal cells throughout the epithelium in all most all the cases of OKC, maximum of about seven cases (46.67%) between the range of 25 and 50%, whereas only two cases (13.33%) of dentigerous cysts showed positivity for bcl-2 immunoreactivity between the ranges of 25 and 50%. But in almost nine cases (60%) of RCs, the bcl-2 immunoreactivity was less than 5%.

Suzuki T et al., in 2005 conducted an analysis in 19 RCs using apoptotic-related factors. The expression of bax was slightly greater than that of bcl-2. The data suggested that apoptotic-related factors are involved in the pathophysiological activity of periapical inflammatory lesions. Such factors may be affected by the structure of lining epithelium and the degree of inflammatory change. [21]

In 2005, Edamatsu [22]et al., examined the expression of Fas, bcl-2, Ki-67, and single-stranded DNA (ssDNA) in dental follicles and dentigerous cysts to clarify the possible role of these apoptosis-related factors in the pathogenesis of dentigerous cysts and in follicles. They found the expression of bcl-2 was weak in epithelial cells neighboring the basement membrane and their expression was lower in follicles than cysts. Eisuke Kichi et al., in 2005 investigated cellular proliferation, cell death, and expression of apoptosis-related proteins in the lining cells of OKCs and DCs. The results showed that Ki-67 positivity was approximately 19% in the whole layer of the lining epithelium of OKC and only 7% in DCs. The bcl-2-positive ratio in the entire layer of the lining epithelium was approximately 32% in OKC and 1% in DC. [23]

Our study also revealed weak expression of bcl-2 in DCs. Only two cases (13.33%) showed their expression between 25 and 50% and three cases (20%) between 10 and 24%. In RCs, nine cases (60%) showed bcl-2 expression in less than 5% of cells.

Conclusions

The bcl-2 positivity of the basal layer of OKC could point to an abnormal control of the cell cycle. The bcl-2 protein over expression could then produce an increase in the survival of the epithelial cells, which results in aggressive growth pattern of OKC. Moreover, the bcl-2 staining can be useful to differentiate OKC from other odontogenic cysts based on the percentage of the cells showing its expression, that being higher in OKC compared to other cysts.

This could be one of the possible reasons for reclassifying OKC as keratinizing cystic odontogenic tumor (KCOT). Further investigation on the distribution of bcl-2-positive cells, either by evaluating primary and recurrent cysts or simple and syndromic OKCs would increase our knowledge on its clinical and pathological behavior.